(1) Field of the Invention
The present invention generally relates to the characterization of motile cells and invasive cells of tumors. More specifically, the invention is directed to methods of isolating motile cells, in particular invasive cells, and the characterization of gene expression in those cells.
(2) Description of the Related Art
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Understanding how cancer cells spread from the primary tumor is important for improving diagnostic, prognostic and therapeutic approaches that allow control of cancer metastasis. Alterations in gene expression along with protein activation by cancer cells leads to transformation, proliferation, invasion, intravasation, dissemination in blood or lymphatic vessels and eventually growth of distant metastases. In order for a tumor cell to become metastatic, it must be able to survive in the circulation and respond appropriately to new environments. This includes being able to migrate both within and beyond the primary tumor, in and out of blood and lymph vessels, and to utilize growth factors available at the site of metastasis for attachment and growth (Lin and Van Golen, 2004).
We have studied the motility-associated behavior of metastatic and non-metastatic mammary tumor cell lines by intravital imaging within primary tumors (Farina et al., 1998a; Wang et al., 2002; Wyckoff et al., 2000a). These studies have shown that the metastatic cells migrate to blood vessels and intravasate in a series of steps that involve active cell motility and may involve chemotaxis (Wang et al., 2002; Wyckoff et al., 2000a; Condeelis and Segall, 2003).
Many of the formative steps that determine the invasive and metastatic potential of carcinoma cells occur within the primary tumor. Much evidence suggests that the progress of cells from normal to invasive and then to metastatic involves progressive transformation through multiple genetic alterations selected by the tumor microenvironment (Hanahan and Weinberg, 2000). To identify the steps in progression and the genes involved in metastasis, recent emphasis has been on the use of molecular arrays to identify expression signatures in whole tumors with differing metastatic potential (Liotta and Kohn, 2001). A well recognized problem here is that primary tumors show extensive variation in properties with different regions of the tumor having different growth, histology, and metastatic potential and where only a small subset of cells within the parental tumor population may be capable of metastasizing (Fidler and Kripke, 1977). The array data derived from whole tumors results inevitably in averaging of the expression of different cell types from all of these diverse regions. The expression signature of invasive tumor cells, arguably the population essential for metastasis, may be masked or even lost because of the contribution of surrounding cells which represent the bulk of the tumor mass. Even so, recent studies of expression profiling of primary tumors suggest that the metastatic potential of tumors is encoded in the bulk of a primary tumor, thus challenging the notion that metastases arise from rare cells within a primary tumor acquired late during tumor progression (Ramaswamy et al., 2003).
This leaves us with a conundrum concerning the contribution of rare cells to the metastatic phenotype. The relative contribution of subpopulations of cells to the invasive and metastatic phenotype of primary tumors has not been assessed due to the difficulty in isolating phenotypically distinct cell populations from whole tumors. In addition, the metastatic cascade has been studied most heavily at the level of extravasation and beyond using experimental metastasis models removing the primary tumor from scrutiny. Thus, the microenvironment of the primary tumor that contributes to invasion and intravasation, and the process of selection of metastatic cells, has not been studied directly (Chambers et al., 2002).
In this context it has become important to develop technologies to separate pure populations of invasive cancer cells for gene expression studies. To this end, the development of Laser Capture Microdissection (LCM) has been an important advance (Bonner et al., 1997). However, the identification of cells within the tumor relies on morphology within fixed tissue making uncertain the identity of the collected cells and their behavior within the tumor before fixation. Alternative approaches involve the collection of cells from metastatic tumors and their expansion in culture (Clark et al., 2000; Kang et al., 2003; Ree et al., 2002). The pitfall of these approaches is that during culturing, the gene expression patterns may change to represent the in vitro culture conditions which are likely to be irrelevant to invasion in vivo.